Da Rocha Lopes, L; (2015) Genetic determinants and genotype-phenotype associations in hypertrophic cardiomyopathy - Contribution of a high-throughput sequencing approach. Doctoral thesis , UCL (University College London).

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Abstract

Background and Aims The application of hypertrophic cardiomyopathy (HCM) genetics in clinical practice has been limited by an incomplete knowledge of the genetic background and a poor understanding of genotype-phenotype relationships. The aims of this study were to study genotype-phenotype relationships in HCM, expand the knowledge on the genetic architecture of the disease, explore genetic modifiers of the phenotype and develop a methodology for interpretation of variants detected by high-throughput sequencing platforms. Methods The study population consisted in consecutive and unrelated HCM patients. In order to analyze coding, intronic and regulatory regions of 41 cardiovascular genes, solution-based sequence capture was followed by massive parallel resequencing. Single-nucleotide variants, small insertion/deletions and copy number variants (CNVs) were called. For the analysis of variants in the coding region, rare, non-synonymous, loss-of-function and splice-site variants were defined as candidates. These variants were tested for associations with clinical phenotype and survival. For the analysis of non-coding variation, variants located in known transcription factor (TF) binding sites and 3’UTR miRNA targets were identified. The performance of an insilico pathogenicity prediction strategy incorporating structural features was explored for MYH7 variants. Selected sarcomere-negative families were studied by whole-exome sequencing (WES). Results Eight-hundred-and-seventy-four patients (49.6±15.4 years, 67.8% males) were studied; likely disease-causing sarcomere protein (SP) gene variants were detected in 383 (43.8%). Patients with SP variants were characterized by younger age and higher prevalence of family history of HCM, family history of sudden cardiac death, asymmetric septal hypertrophy, higher maximal wall thickness (P-values<0.0005) and increased cardiovascular death (P-value=0.012). Similar associations were observed for individual SP genes. Patients with ANK2 variants had greater maximum wall thickness (P-value=0.0005). Associations at a lower level of significance were demonstrated with variation in other non-SP genes. Four CNVs were detected in MYBPC3, PDLIM3, TNNT2 and LMNA. Fourteen percent carried non-coding variants mapping to TF binding sites. The pathogenicity prediction for MYH7 missense variants had an accuracy of 0.93; the phenotype predictor had an accuracy of 0.79 and novel genotype-phenotype associations were suggested. Three families were studied using WES; in two of these families the main candidate variants were in OBSCN and TTN. Conclusions Patients with SP variants differed from those without with respect to age, family history, ventricular morphology and prognosis. Novel associations were demonstrated between individual sarcomeric genes and several phenotype traits and for the first time, associations between rare variants in non-SP genes and phenotype were described. CNVs and non-coding variation in SP genes can additionally contribute to the genetic architecture of HCM. Pathogenicity prediction incorporating structural features revealed additional genotype-phenotype associations. Whole-exome sequencing suggested new causal genes.

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